Lw. Mitchell et al., THE PHYLOGENETICALLY CONSERVED HISTIDINES OF ESCHERICHIA-COLI PORPHOBILINOGEN SYNTHASE ARE NOT REQUIRED FOR CATALYSIS, The Journal of biological chemistry, 270(41), 1995, pp. 24054-24059
Porphobilinogen synthase (PEGS) is a metalloenzyme that catalyzes the
first common step of tetrapyrrole biosynthesis, the asymmetric condens
ation of two molecules of 5-aminolevulinic acid (ALA) to form porphobi
linogen. Chemical modification data implicate histidine as a catalytic
residue of PBGS from both plants and mammals. Histidine may participa
te in the abstraction of two non-ionizable protons from each substrate
molecule at the active site. Only one histidine is species-invariant
among 17 known sequences of PBGS which have high overall sequence simi
larity. In Escherichia coli PBGS, this histidine is His(128). We perfo
rmed site-directed mutagenesis on His(128), replacing it with alanine.
The mutant protein H128A is catalytically active. His(128) is part of
a histidine- and cysteine-rich region of the sequence that is implica
ted in metal binding. The apparent K-d for Zn(II) binding to H128A is
about an order of magnitude higher than for the wild type protein. E.
coli PBGS also contains His(126) which is conserved through the mammal
ian, fungal, and some bacterial PBGS. We mutated His(126) to, alanine,
and both His(126) and His(128) simultaneously to alanine, All mutant
proteins are catalytically competent; the V-max values for H128A (44 u
nits/mg), H126A (75 units/mg), and H126/128A (61 units/mg) were simila
r to wild type PBGS (50 units/mg) in the presence of saturating concen
trations of metal ions. The apparent K-d for Zn(II) of H126A and H126/
128A is not appreciably different from wild type. The activity of wild
type and mutant proteins are all stimulated by an allosteric Mg(II);
the mutant proteins all have a reduced affinity for Mg(II). We observe
a pK(alpha) of similar to 7.5 in the wild type PBGS k(cat)/K-m pH pro
file as well as in those of H128A and H126/128A, suggesting that this
pK(alpha) is not the result of protonatioaldeprotonation of one of the
se histidines. H128A and H126/128A have a significantly increased K-m
value for the substrate ALA. This is consistent with a role for one or
both of these histidines as a ligand to the required Zn(II) of E. col
i PBGS, which is known to participate in substrate binding. Past chemi
cal modification may have inactivated the PBGS by blocking Zn(II) and
ALA binding. In addition, the decreased K-m for E. coli PBGS at basic
pH allows for the quantitation of active sites at four per octamer.